The objective of this proposal is to investigate the substrate binding, isoforms, associated proteins, and substrate size specificity of E.C.3.4.24.15 (EP24.15), with the goal of availing design of new therapeutic drugs and small molecules, to modulate development of neuroendocrine/neurodegenerative diseases and endocrine cancer(s). EP24.15 is the prototypic member of the metalloendopeptidase family, and is widely expressed in the brain, pituitary, and reproductive organs. Extracellularly, EP24.15 cleaves and modulates neuropeptides, and thus EP24.15 plays a significant role in neuroendocrine hormone regulation, pain perception, development of neurodegenerative diseases, and prostate cancer cell growth. The goal of this proposal is to further the understanding of the functions of EP24.15, of its isoforms, and of closely related metalloendopeptidases, with the goal of understanding the prominence of EP24.15 in neuroendocrine/neurodegenerative diseases and cancer. Therefore, this proposal will integrate biochemical, genetic, structural, and proteomic approaches to study substrate binding, isoforms, associated proteins, and substrate size specificity of EP24.15. Therefore, the specific aims are: SPECIFIC AIM 1. Elucidate functional sites conferring substrate binding and catalysis. Aim la. Determine which residues comprise the substrate (inhibitor) binding site of EP24.15. Aim Ib. Determine which residues are crucial for substrate catalysis and specificity. SPECIFIC AIM 2. Determine isoforms and protein binding partners of EP24.15 and of related enzymes. Aim 2a. Identify the extracellular and intracellular isoforms of EP24.15 and EP24.16. Aim 2b. Characterize complexes of EP24.15 with associated protein-binding partners. SPECIFIC AIM 3. Unveil new substrates and mechanism of substrate size selectivity. Aim 3a. Determine what other (neuro)peptide substrates exist for EP24.15. Aim 3b. Elucidate structural changes upon substrate binding, focusing on domain movements. [unreadable] [unreadable]